An asymmetry in the synthesis of leading and lagging DNA strands creates the “end problem” for replication of linear genomes. .sup.1 To overcome this problem, eukaryotic chromosomes have specialized end structures, telomeres, consisting of TTAGGG repeats. .sup.2 Telomerase.sup.3,4 is a ribonucleoprotein enzyme that elongates telomeres and therefore maintains chromosomal stability in majority of cancer cells during cell doubling. .sup.5 The gradual loss of DNA from the ends of telomeres during cell doubling has been implicated in the control of cellular proliferative potential in somatic cells. .sup.6
Normal cultured human cells have a limited replication potential in culture. Normal cells in culture replicate until they reach a discrete point at which population growth ceases. This is termed mortality stage 1 (M1 stage) and is caused by the shortening of a few telomeres to a size that leads to a growth arrest called cellular senescence. This stage can be bypassed by abrogation of the function of p53 and pRB human tumor suppressor genes. The cells then can continue to proliferate with further decreases in telomere length until another check point termed mortality stage 2 (M2 stage) or crisis stage. The growth arrest in the M2 stage is caused by balance between the cell proliferation and cell death rate. At this stage, when most of the telomeres are extremely short, end-to-end fusions and chromosomal breakage-fusion cause marked chromosomal abnormalities and apoptosis. Under rare circumstances, a cell can escape M2 and become immortal by stabilizing the length of its telomeres. This occurs through the activation of the enzyme telomerase or an alternative mechanism of telomere lengthening (ALT). .sup.7,8
Human germline.sup.9 and the majority of cancer cells.sup.3 express telomerase. Telomerase is a ribonucleoprotein enzyme that elongates telomeres and, therefore, maintains chromosomal stability in majority of cancer cells during cell doubling. .sup.10 Indeed, elongation of shortened telomeres by telomerase is a major mechanism of telomere maintenance in the human cancer cells. Inhibition of telomerase limits the growth of human telomerase positive cancer cells.sup.11 by decreasing telomere length.
Elongation of shortened telomeres by telomerase is a well known mechanism of telomere maintenance in the human cancer cells. From a biological point of view, telomerase elongates telomeres by the addition of repetitive DNA sequences of the TTAGGG-type (telomeric sequences), at the end of the telomere, during cell division. Through this action, telomerase imparts chromosomal stability and renders the cell immortal. In attempting to obtain selective inhibitors as useful tools for studying this enzyme, inhibitory effects of nucleotide analogues have been investigated in cell-free systems (Yamaguchi et al., (2001, Nucleic Acids Research Supplement No. 1 211-212). Since proliferating cells including cancer cells express telomerase activity while normal human somatic cells do not express telomerase activity at levels sufficient to maintain telomere length over many cell divisions as seen in cancer cells, telomerase is a good target for treating proliferative disorders including cancer.
Currently, strategies aimed at selectively treating the cancers from telomerase positive cells involve modulation of TERT (Telomerase Reverse Transcriptase) function or length of telomeres by antisense strategy, dominant negative mutants or pharmacological agents (see, Bisoffi et al., Eur J Cancer, 1998, 34: 1242-1249; Roth et al., Leukemia, 2003, 17:2410-2417; Damm et al., EMBO J., 2001, 20:6958-6968; U.S. Pat. Nos. 6,294,332, 6,194,206, 6,156,763 and 6,046,307). The use of nucleoside analogs (e.g., AZT) has been attempted to interfere with human telomerase activity with an aim to treat cancers. The methods disclosed in the prior art administering nucleoside analogs to modify telomerase activity, however, are not satisfactory or are not suitable in a clinical setting because their clinical utility is limited by a low therapeutic ratio, i.e., the ratio of toxic dose to effective dose.
Prolonged exposure of telomerase positive cell lines to AZT failed to induce any significant telomere shortening at a concentration of the drug equal to 100 .mu.M (Murakami, J., Nagai. N., Shigemasa. K., Ohama. K. Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitor in gynaecological cancer cell lines. Eur. J. Cancer 35, 1027-1034 (1999) or even 800 .mu.M (Gomez D E, Tejera A M, Olivero O A. Irreversible telomere shortening by 3′-azido-2′,3′-dideoxythymidine (AZT) treatment. Biochem Biophys Res Commun. 1998; 246(1): 107-10; Tejera A M, Alonso D F, Gomez D E, Olivero O A. Chronic in vitro exposure to 3′-azido-2′,3′-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells. Breast Cancer Res Treat. 2001; 65(2):93-9).
While peak serum concentration after taking single oral dose of 300 mg of AZT was less than 10 .mu.M, and it was rapidly absorbed within 0.5 h (Morse G D, Olson J, Portnore A, Taylor C, Plank C, Reichman R C Pharmacokinetics of orally administered zidovudine among patients with hemophilia and asymptomatic human immunodeficiency virus (HIV) infection. Antiviral Res. 1989 March; 11(2):57-65). Standard AZT treatment is 500 or 600 mg/day in two or three divided doses for adults according to the recommendations of the manufacturer of Retrovir™ (AZT). It has been reported that even a short-time exposure to AZT at a concentration of 5 .mu.M induces undesirable toxic effects on mammalian cells in vitro and in vivo (Roskrow M, Wickramasinghe S N. Acute effects of 3′-azido-3′-deoxythymidine on the cell cycle of HL60 cells. Clin Lab Haematol. 1990; 12(2):177-84.). Based on these reports, one can predict that doses of nucleoside analogs such as AZT high enough to provide antitelomerase and antitumor efficacies can be highly toxic and cause damage to important tissues in humans. Thus, there is need for the identification of therapeutic nucleoside analogs, which have modulation or inhibitory activity against human telomerase, and development of methods of treatment of cancers in which telomerase contributes to the immortality and undesirable proliferation.